The invention relates to the use of amidopyrylium compounds as labeling groups in procedures for the detection of analytes, and to novel amidopyrylium compounds.
Xanthenes are long-known and well-researched fluorescent dyes. A related dye having a modified parent structure, the 3,10-bis(dimethylamino)-5-methyl-6-oxo-6H[1]-benzopyrano[3,2-c]quinolinium cation, has been described by H. Harnisch, Liebigs Ann. Chem. 751, 155-158 (1971).
On account of their similarity to pyrans, compounds of this type were also designated as amidopyrylium compounds by K. H. Drexhage, Structure and Properties of Laser Dyes, in: F. P. Schxc3xa4fer, Topics in Applied Physics, Vol. I, Dye Lasers, Springer-Verlag, Berlin, Heidelberg, N.Y., 1973. In this publication, amidopyrylium compounds are further designated as dye 140 and 141. Information on the use of amidopyrylium compounds as fluorescent labeling groups in analysis is not found in the literature.
Fluorescent dyes used hitherto in chemical, medical and biological analysis mostly absorb in the range  less than 600 nm. Serious disadvantages result therefrom when using them as a labeling group, in particular in biological systems: For diagnostic systems, it is expedient to be able to employ inexpensive light sources such as, for example, laser diodes (635 or 680 nm) or helium-neon lasers (633 nm). In order to guarantee an effective excitation of a fluorescent dye, its absorption maximum should if possible be in the vicinity of the emission wavelength of the light source used. However, this is often not conferred in the case of the known dyes. Furthermore, in many cases the absorption spectra of the known dyes overlap with the absorption of fluorescent substances from biological systems. It is therefore desirable to make available fluorescent dyes without the disadvantages mentioned for a reliable and precise detection of an analyte in a biological system.
For use as a labeling group in detection procedures for analytes, in addition to a simple and reliable detectability, a good solubility in various solvents, in particular in aqueous systems, is necessary. Furthermore, compounds of this type should be simple and inexpensive to prepare and have a good durability, i.e. shelf life.
An object of the present invention was thus to make available suitable fluorescent dyes which can be employed as labeling groups for procedures for the detection of analytes, which in particular have absorption maxima which allow the use of inexpensive light sources, absorb outside the absorption range of substances contained in biological samples, exhibit good solubility or/and are distinguished by a high fluorescence quantum yield, in order at least partially to avoid the disadvantages of the prior art.
This object has been achieved by the use of compounds of the general formula I 
as labeling groups in a procedure for the detection of an analyte, where
Y is oxygen or Nxe2x80x94R8,
R1, R2, R3 and R8, on each occurrence, are independently hydrogen, a phenyl group, a phenylalkyl group having 1-3 C atoms in the alkyl chain, a polyether group or a hydrocarbon group having up to 20 C atoms, preferably having up to 6 C atoms, which can optionally contain one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, carboxyl, carbonyl, alkoxy or/and alkoxycarbonyl groups, or one or more of the radicals R1, R2, R3 and R8 form a ring system with an adjacent substituent, R4, R5, R6 and R7 in each case independently of one another are hydrogen, halogen, phenyl, a hydroxyl, amino, sulfo or carboxyl group or a hydrocarbon group having up to 15 C atoms, where the hydrocarbon groups can comprise alkyl, alkenyl, alkynyl, cycloalkyl, aryl or/and heteroaryl radicals and can optionally in each case contain one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, carboxyl, aldehyde, alkoxy or/and alkoxycarbonyl groups,
where one or more of the radicals R4, R6 and R7 can form a ring system with an adjacent substituent,
Cyc is an oroanic radical which comprises a ring system selected from aromatic, heteroaromatic, quinoid or/and cycloaliphatic rings, which can optionally contain one or more substituents, preferably selected from halogens, amino, hydroxyl, sulfo, carboxyl, aldehyde, alkoxy or/and alkoxycarbonyl groups, and
X is optionally anions present for charge equalization.
The compounds of the general formula I can be employed as labeling groups in procedures for the qualitative or/and quantitative determination of an analyte. The determination can be carried out in aqueous liquids, e.g. samples of body fluids such as, for example, blood, serum, plasma or urine, waste water samples or foodstuffs. The procedure can also be carried out as a wet test, e.g. in a cuvette, or as a dry test in an appropriate reagent carrier. The determination of the analytes can be carried out here by means of a single reaction or by means of a sequence of reactions.
Surprisingly, the use of compounds of the general formula I showed very good results in chemical and in particular in medical and biological detection procedures for the determination of an analyte.
The compounds of the general formula I can be used in all known chemical, medical and biological detection procedures in which fluorescent dyes are suitable as labeling groups. Procedures of this type are known to the person skilled in the art and therefore do not have to be elaborated further.
In a particularly preferred embodiment, the compound of the general formula I is coupled covalently to a receptor specific for the analyte to be detected. The specific receptor is any suitable compound or any suitable molecule, preferably it is a peptide, polypeptide or a nucleic acid. The compounds I or conjugates of this compound can be used, for example, in nucleic acid hybridization procedures or immunochemical procedures. Procedures of this type are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor.
A further object of the present invention was to make available novel amidopyrylium compounds which are suitable in particular for use as labeling groups in procedures for the detection of analytes and at least partially avoid the disadvantages of the prior art.
This object has been achieved by a compound of the general formula I 
where
Y, R1-R7 and Cyc have the meanings indicated in claim 1, X is optionally anions present for charge equalization, with the proviso that if Y is oxygen, R1, R2 and R3 are methyl and R4, R5, R6 and R7 are hydrogen,
Cyc does not have a structure of the formulae II or III 
An advantage of the compounds I is that owing to an almost arbitrary substituent variation the properties of individual compounds, e.g. the spectroscopic properties, the position of the absorption maxima, the solubility properties, the fluorescence decay time and the magnitude of the quantum yield, varies strongly and thus can be selected as desired. In this way, interferences with interfering substances in samples, such as, for example, serum, blood or plasma etc. can be reduced or even avoided completely. The preparation of the compounds I can be carried out by methods known per se in a simple and inexpensive manner, as is explained in the following examples. Furthermore, the compounds can be handled unproblematically. The further advantage of the compounds I is the large Stokes shift of the fluorescence, by means of which a good separation of the excitation radiation is facilitated. The compounds are furthermore distinguished by a high stability, which in particular has a positive effect on their storability.
Preferably, Y is oxygen or/and R5 comprises an aromatic, optionally substituted ring system.
The compounds preferably have a group capable of covalent coupling, e.g. xe2x80x94COOH, xe2x80x94NH2, xe2x80x94OH or/and xe2x80x94SH. By means of this coupling group, the compound can be coupled to a carrier or/and to a biomolecule by methods known per se. The carrier selected can be any suitable material, e.g. porous glass, plastics, ion-exchange resins, dextrans, cellulose, cellulose derivatives or/and hydrophilic polymers. The biomolecules are preferably selected from peptides, polypeptides, nucleotides, nucleosides, nucleic acids, nucleic acid analogs or/and haptens.
Surprisingly, the absorption maximum and the fluorescence quantum yield are not significantly changed by coupling of the compounds according to the invention to the abovementioned carriers and biomolecules.
In a preferred class of compounds, R1 is bridged with R7 or/and R2 with R4 and form a ring system, in particular having 5- or 6-membered rings. In a particularly preferred class of compounds, Cyc in formula I has a structure of the formulae IV, V or VI 
where R1xe2x80x2, R2xe2x80x2 and R3xe2x80x2 are defined as R1, R2 and R3 above and R9-R11 are defined as R4-R7 above.
In a further preferred class of compounds, R1xe2x80x2 is bridged with R11 or/and R2xe2x80x2 with R10 and form a ring system, in particular a 5- or 6-membered ring.
Examples of particularly preferred classes of compounds are shown in the general formulae VIIa to f: 
where
the dashed lines are optionally double bonds, in whose presence the radicals R bonded via a dashed line are absent,
X, Y, R1, R2, R3, R1xe2x80x2, R2xe2x80x2 and R3xe2x80x2 are as defined above, and R is in each case independently defined as R4 above.
Actual examples of compounds according to the invention are shown in table 1 below.